Migration of noble gas tracers at the site of an underground nuclear explosion at the Nevada National Security Site.

As part of an underground gas migration study, two radioactive noble gases (37Ar and 127Xe) and two stable tracer gases (SF6 and PFDMCH) were injected into a historic nuclear explosion test chimney and allowed to migrate naturally. The purpose of this experiment was to provide a bounding case (natural transport) for the flow of radioactive noble gases following an underground nuclear explosion. To accomplish this, soil gas samples were collected from a series of boreholes and a range of depths from the shallow subsurface (3 m) to deeper levels (~160 m) over a period of eleven months. These samples have provided insights into the development and evolution of the subsurface plume and constrained the relative migration rates of the radioactive and stable gas species in the case when the driving pressure from the cavity is low. Analysis of the samples concluded that the stable tracer SF6 was consistently enriched in the subsurface samples relative to the radiotracer 127Xe, but the ratios of SF6 and 37Ar remained similar throughout the samples.

Source term estimation of radioxenon released from the Fukushima Dai-ichi nuclear reactors using measured air concentrations and atmospheric transport modeling.

Systems designed to monitor airborne radionuclides released from underground nuclear explosions detected radioactive fallout across the northern hemisphere resulting from the Fukushima Dai-ichi Nuclear Power Plant accident in March 2011. Sampling data from multiple International Modeling System locations are combined with atmospheric transport modeling to estimate the magnitude and time sequence of releases of (133)Xe. Modeled dilution factors at five different detection locations were combined with 57 atmospheric concentration measurements of (133)Xe taken from March 18 to March 23 to estimate the source term. This analysis suggests that 92% of the 1.24 × 10(19) Bq of (133)Xe present in the three operating reactors at the time of the earthquake was released to the atmosphere over a 3 d period. An uncertainty analysis bounds the release estimates to 54-129% of available (133)Xe inventory.

Offset prediction for charge-balanced stimulus waveforms.

Functional electrical stimulation with cuff electrodes involves the controlled injection of current into an electrically excitable tissue for sensory or motor rehabilitation. Some charge injected during stimulation is 'lost' at the electrode-electrolyte interface when the charge carrier is translated from an electron to an ion in the solution. The process of charge injection through chemical reactions can reduce electrode longevity and implant biocompatibility. Conventionally, the excess charge is minimized by complex hardware solutions, which are often not appropriate for robust long-term implantable solutions. Here, we present a method of waveform design that minimizes irrecoverable charge during continuous pulsing through the use of biphasic waveforms with unequally charged phases. We developed an equivalent electrical model of the electrode-electrolyte impedance based on the electrode's surface chemistry during psuedo-bipolar stimulation conditions. Simulations with the equivalent circuit determined the uncompensated charge to be a function of stimulus parameters. In vitro stimulation experiments in saline confirmed that we could preemptively compensate for the excess charge following biphasic stimulus waveforms. As a result, there was a 92% reduction in the pre-pulse potential after a pulse train with this new waveform design when compared to stimulation with conventional biphasic waveforms.

Elevated radioxenon detected remotely following the Fukushima nuclear accident.

We report on the first measurements of short-lived gaseous fission products detected outside of Japan following the Fukushima nuclear releases, which occurred after a 9.0 magnitude earthquake and tsunami on March 11, 2011. The measurements were conducted at the Pacific Northwest National Laboratory (PNNL), (46°16'47″N, 119°16'53″W) located more than 7000 km from the emission point in Fukushima Japan (37°25'17″N, 141°1'57″E). First detections of (133)Xe were made starting early March 16, only four days following the earthquake. Maximum concentrations of (133)Xe were in excess of 40 Bq/m(3), which is more than ×40,000 the average concentration of this isotope is this part of the United States.

Evaluation of the OSOM Trichomonas rapid test versus wet preparation examination for detection of Trichomonas vaginalis vaginitis in specimens from women with a low prevalence of infection.

The OSOM Trichomonas rapid test (OSOM Trich) was compared to the wet preparation examination (WP) for the detection of Trichomonas vaginalis vaginitis in women with a low prevalence of infection. A total of 19/1,009 (2%) women had T. vaginalis infection. OSOM Trich had very good performance, with sensitivity, specificity, efficiency, positive predictive value, and negative predictive value of 94.7, 100, 99.9, 100, and 99.9%, respectively. The implementation of OSOM Trich would decrease labor costs.

Social ecological predictors of prostate-specific antigen blood test and digital rectal examination in black American men.

BACKGROUND: Black American men continue to suffer disproportionately from epidemically higher rates of prostate cancer. We hypothesize that complex reasons for persistently higher death rates of prostate cancer in this group are steeped in social factors associated with health access. METHODS: We utilized data from the It's All About U prostate cancer prevention study among black men to investigate: 1) what social ecological factors were predictive of prostate-specific antigen (PSA) testing and digital rectal examinations (DRE); 2) if black men were aware of prostate cancer screening and, if screening was available, would they take the PSA and DRE? Quantitative cross-sectional data from a cohort of 276 black men with no diagnosis of prostate cancer were analyzed to identify characteristics, beliefs, practices and attitudes of this group toward prostate cancer screening. We created a social ecological model to examine which social factors (i.e., environmental, personal, person/environment interplay, black culture and institutional policy) were predictive of PSA and DRE, PSA only and DRE only. To reduce data and identify data patterns, factor analyses (tested for reliability by calculating Cronbach alpha scores) were performed. Variables were standardized with Z scores and analyzed with predictive analytic software technology (SPSS, version 12). A multivariate binary logistic regression was conducted to identify predictors of PSA and DRE. RESULTS: A significant predictor of both PSA and DRE was the physician's direct prostate cancer communication message (P<0.010). Significant correlations exist in PSA and DRE outcomes with a physician's engaging communication style (P<0.012), encouragement to screen (P<0.001) and sharing prostate cancer information (P<0.001); as was men understanding the serious risk of prostate cancer (P<0.001), culture (P<0.004), positive interaction with healthcare staff, significant other(s) and providers (P<0.001), and environmental dimensions (P<0.006). A profile of four major self-reported barriers to screening were identified (i.e., fear, internal locus of health, comfort level and external locus of health). Lastly, men who utilized health systems with a prostate cancer screening policy had high percentages of PSA and DRE (63.3%), PSA only (70.9%) and DRE only (81.7%). CONCLUSION: A physician's aggressive, positive engagement in shared decision-making, tailored social influences promoting prostate cancer prevention among black men, as well as institutional screening policy, has the potential to increase early detection and reduce morbidity among this group.

Culture, black men, and prostate cancer: what is reality?

BACKGROUND: The worldwide incidence of prostate cancer is higher among American black men than any other male group. In the United States, lack of participation in screening for prostate cancer by black men is influenced by several cultural factors, including knowledge, health beliefs, barriers, and relationships with primary healthcare providers. METHODS: We used the qualitative and paralleling descriptive quantitative findings of a mixed-method longitudinal study exploring prostate cancer screening behaviors among 277 black men. RESULTS: Five themes were identified as critical elements affecting men's screening for prostate cancer: lack of knowledge, communication, social support, quality of care, and sexuality. These themes were associated with a sense of disconnectedness by black men from the healthcare system and contributed to nonparticipation in prostate cancer early detection activities. CONCLUSIONS: Lack of discussion about the decision to screen for prostate cancer and general lack of culturally appropriate communication with healthcare providers has engendered distrust, created fear, fostered disconnect, and increased the likelihood of nonparticipation in prostate cancer screening among black men.

Recruiting Black/African American men for research on prostate cancer prevention.

BACKGROUND: Black/African American men die of prostate cancer at a greater rate relative to other males. During the period from 1992 to 1998, prostate cancer incidence rates in the United States were 234.2 per 100,000 persons among non-Hispanic black males and 144.6 per 100,000 persons among white males. The reasons for these increased rates of prostate cancer among black males are largely unknown, but increased mortality is associated with late detection. The authors conducted a longitudinal study of black men that investigated prostate cancer prevention behaviors within this population. The purpose of the current article is to identify successful recruitment strategies that were reported by participants in this study of prevention behaviors. METHODS: Qualitative research methods were used to elucidate men's thoughts, attitudes, beliefs, and practices regarding prostate cancer prevention behaviors and to identify strategies for attracting black men to research programs and retaining them in these programs. RESULTS: Ethnocentric recruitment strategies that were identified included the development of tailored printed materials; the use of targeted locations; and a personalized, participatory approach for engaging potential participants. We contacted 498 black men and enrolled a cohort of 277 non-Hispanic black males (75% of whom were recruited within a 9-week period) in the current study. CONCLUSIONS: Unlike other studies that reported difficulty in recruiting African American men, the current study did not encounter such difficulties. The authors attribute their success to culturally attractive Afrocentric materials; cultural sensitivity; a caring, professional, personalized ethnic approach; respect; and participatory involvement of the target population. Nonetheless, the authors did encounter barriers, such as lack of physician interest and lack of trust in quality medical care. These barriers must be overcome before black males can be engaged and retained in research studies on prostate cancer prevention.

Identification of anti-thrombin antibodies in the antiphospholipid syndrome that interfere with the inactivation of thrombin by antithrombin.

The combined presence of anti-phospholipid (PL) Ab, including lupus anticoagulants (LAC) and/or anticardiolipin Ab (aCL), and thrombosis is recognized as the antiphospholipid syndrome (APS). LAC are detected as an inhibitory effect on PL-restricted in vitro blood coagulation tests, and are comprised mainly of Ab against beta(2) glycoprotein I and prothrombin (PT). Recently, anti-PT Ab (aPT) were found to be associated with thrombosis by some investigators, although this is not confirmed by others. Considering that aPT are heterogeneous in patients and that PT is converted into thrombin, we hypothesize that certain aPT in patients may bind to thrombin, and that some of such anti-thrombin Ab may interfere with thrombin-antithrombin (AT) interaction and thus reduce the AT inactivation of thrombin. To test this hypothesis, we searched for anti-thrombin Ab in APS patients and then studied those found for their effects on the AT inactivation of thrombin. The results revealed that most, but not all, aPT-positive patient plasma samples contained anti-thrombin Ab. To study the functional significance of these Ab, we identified six patient-derived mAb that bound to both PT and thrombin. Of these mAb, three could reduce the AT inactivation of thrombin, whereas others had minimal effect. These findings indicate that some aPT in patients react with thrombin, and that some of such anti-thrombin Ab could inhibit feedback regulation of thrombin. Because the latter anti-thrombin Ab are likely to promote clotting, it will be important to develop specific assays for such Ab and study their roles in thrombosis in APS patients.

Anticardiolipin antibodies from patients with the antiphospholipid antibody syndrome recognize epitopes in both beta(2)-glycoprotein 1 and oxidized low-density lipoprotein.

BACKGROUND: We recently suggested that many anticardiolipin antibodies bind only to oxidized cardiolipin (OxCL) and/or to OxCL-beta(2)-glycoprotein 1 (beta(2)GP1) adducts but not to a "reduced" cardiolipin that is unable to undergo oxidation. To test this hypothesis, we investigated 24 sera, 4 protein A-purified IgG fractions, and 3 human monoclonal antibodies that were all isolated from patients with antiphospholipid antibody syndrome (APS); testing was also performed in 7 controls. Two monoclonal antibodies (IS3 and IS4) were selected for binding to CL and one was selected for binding to beta(2)GP1 (LJB8). METHODS AND RESULTS: By chemiluminescent immunoassay, all APS sera samples bound only to OxCL and not to reduced CL, and the binding was inhibited >95% by OxCL but not reduced CL. All purified IgG fractions bound to beta(2)GP1 but only when the beta(2)GP1 was plated on microtiter wells coated with OxCL. All 3 monoclonal antibodies bound only to OxCL. On Western blots, IS4 and LJB8 bound to beta(2)GP1 as well as to delipidated apoB of oxidized LDL but not to native apoB. IS3 also bound to oxidized apoB on Western blot. Covalent modification of beta(2)GP1 with oxidation products of CL made it more antigenic for APS serum samples, for purified IgG fractions, and for the monoclonal antibodies. CONCLUSIONS: These data support the hypothesis that oxidation of CL is needed to generate epitopes for many anticardiolipin antibodies and that some of these epitopes are covalent adducts of OxCL with beta(2)GP1 or apoB.

Propranolol affects stress-induced leukocytosis and cellular adhesion molecule expression.

In this study, the impact of the beta-adrenergic antagonist propranolol on resting and acute psychological- and physical-stress-induced circulating leukocyte numbers and the density of cellular adhesion molecules was investigated. In a randomized double-blind crossover design, 45 healthy volunteers performed a 15-min public speaking task and 21 subjects performed a 16-min bicycle exercise after 5 days of ingesting a placebo and after 5 days of ingesting 100 mg/day propranolol. One week of ingesting propranolol modestly elevated the numbers of CD62L+ (P<0.019) but not CD62L- T-lymphocytes. Moreover, propranolol preferentially blunted-psychological stress-induced increases in naïve T-helper (CD4+CD62L+; P<0.049) and naïve T-cytotoxic lymphocytes (CD8+CD62L+; P<0.003), as well as activated T-cytotoxic lymphocytes (CD8+CD29+; P<0.005). However, exercise-induced increases in leukocyte numbers were enhanced following propranolol treatment (P<0.04). In contrast to the effect on the numbers of adhesion-molecule-bearing cells, there was only a modest effect of propranolol on stress-induced alterations of the density of CD62L, CD54 and CD11a. In this study, propranolol treatment interfered with the adrenergic regulation of circulating leukocyte numbers by blunting psychological stress effects but enhancing exercise effects. Propranolol affected the cell activation status to a lesser extent, as reflected by the density of adhesion molecules.

High resolution, high-throughput amide deuterium exchange-mass spectrometry (DXMS) determination of protein binding site structure and dynamics: utility in pharmaceutical design.

Mass spectrometry-based peptide amide deuterium exchange techniques have proven to be increasingly powerful tools with which protein structure and function can be studied, and are unparalleled in their ability to probe sub-molecular protein dynamics. Despite this promise, the methodology has remained labor-intensive and time consuming, with substantial limitations in comprehensiveness (the extent to which target protein sequence is covered with measurable peptide fragments) and resolution (the degree to which exchange measurements can be ascribed to particular amides). I have developed and integrated a number of improvements to these methodologies into an automated high throughput, high resolution system termed Deuterium Exchange Mass Spectrometry (DXMS). With DXMS, complete sequence coverage and single-amide (amino acid) resolution are now rapidly accomplished. DXMS is designed to work well with large proteins and when only small amounts of material are available for study. Studies can be performed upon a receptor-ligand pair as they exist on or within a living cell (in vivo) without prior purification, allowing effective in situ study of integral membrane protein receptors. We have ambitious initiatives underway to make DXMS widely available both for basic academic research studies and commercial drug discovery efforts. In this paper I present an overview of DXMS technology and highlight some of the benefits it will provide in drug discovery and basic proteomics research.

Peripheral blood mononuclear cell-endothelial adhesion in human hypertension following exercise.

OBJECTIVE: To determine the effects of hypertension and exercise on interleukin-6 (IL-6) levels and mononuclear cell adhesion to endothelial cells. DESIGN: Twelve hypertensive and 33 normotensive volunteers were studied prior to and following exhaustive exercise. End points were stimulated IL-6 levels and peripheral blood mononuclear cell (PBMC) CD11a (LFA-1) expression and in vitro PBMC adhesion to human umbilical venous endothelial cells (HUVEC). RESULTS: In response to exercise, all subjects showed a significant increase in lymphocyte CD11a a density and in IL-6 levels (P < 0.001). Compared to normotensives, hypertensives showed significantly greater mean density of CD11a on lymphocytes (P< 0.05) and on monocytes (P < 0.05). In response to exercise, hypertensive subjects showed a twofold greater increase in IL-6 as compared to normotensives (+ 240 pg/ml versus + 123 pg/ml, respectively; P< 0.05). PBMC adhesion to HUVEC was increased in hypertensives but decreased in normotensives following exercise (P< 0.03). CONCLUSION: The findings suggest that exercise leads to increased mononuclear cell adhesion to endothelial cells in patients with hypertension, possibly through cytokine-induced activation of mononuclear cell CD11a. These findings, coupled with prior data indicating increased endothelial activation in hypertension, may be relevant to the increased risk of atherosclerosis in human hypertension.

An IgG antiprothrombin antibody enhances prothrombin binding to damaged endothelial cells and shortens plasma coagulation times.

OBJECTIVE: To test the hypothesis that some lupus anticoagulants are antiprothrombin antibodies, and that such antibodies enhance prothrombin binding to endothelial cells (EC) and thus promote clotting on the cell surface. METHODS: We generated a monoclonal antiprothrombin antibody (designated IS6) from a patient with primary antiphospholipid syndrome (APS). The antibody was analyzed for its binding properties, lupus anticoagulant activity, and pathophysiologic activity, using an EC-based plasma coagulation assay. RESULTS: IS6 is the first patient-derived monoclonal IgG antiprothrombin antibody. It bound to prothrombin with low affinity, reacted with 3 phospholipids (cardiolipin, phosphatidylethanolamine, and phosphatidylserine), and showed lupus anticoagulant activity. Moreover, IS6 enhanced the binding of prothrombin to damaged EC and shortened the EC-based plasma coagulation times. CONCLUSION: These findings suggest that IS6 may promote coagulation in areas of damaged EC in the host, and thus contribute to thrombosis in patients with APS.

Characterization of IgG monoclonal anti-cardiolipin/anti-beta2GP1 antibodies from two patients with antiphospholipid syndrome reveals three species of antibodies.

Antiphospholipid antibodies (aPL), including antibodies detected in anti-cardiolipin (aCL) enzyme-linked immunosorbent assays and in lupus anticoagulant (LA) tests, are strongly associated with recurrent thrombosis and recurrent fetal loss, i.e. the antiphospholipid syndrome (APS). Although recent studies suggest that most APS-associated aCL are directed against the phospholipid (PL)-binding plasma protein beta2-glycoprotein 1 (beta2GP1), the precise nature of aCL binding specificities remains controversial. To address the issue of aCL specificity we generated five new monoclonal IgG aCL from two patients with APS. Characterization of these five aCL, as well as two previously published IgG aCL, revealed three patterns of reactivity: (1) four antibodies reacted strongly with human beta2GP1-cardiolipin (CL) complexes and weakly with human beta2GP1 alone; (2) two antibodies recognized bovine beta2GP1, but not human beta2GP1; (3) one antibody reacted with complexes of human beta2GP1 and CL, but not with human beta2GP1 alone. Only one monoclonal displayed weak LA activity. These patient-derived IgG monoclonal antibodies, and additional ones to be generated, may help define varying species of antibodies detected in aCL assays and identify the specific antibodies that may be pathogenic.

Estrogen responses in bovine fetal uterine cells involve pathways directed by both estrogen response element and activator protein-1.

Objectives were to examine possible roles of estrogen receptor (ER) in development of the bovine uterine endometrium in the context of ER type, enhancer type, and ligand-independent activation. Expression vectors producing either ERalpha or ERbeta were introduced into fetal uterine cells from Day 110 to 120 of gestation (UBF120 cells) and into rat embryo fibroblasts (Rat-1 cells), neither of which express endogenous ER. Reporter constructs containing either an estrogen response element (ERE) or activator protein-1 (AP-1) response element were cotransfected. These reporters were also transfected into fetal uterine cells from Day 180 to 200 of gestation (UBF180 cells), which express ER. In UBF120 and Rat-1 cells transfected with either ERalpha or ERbeta, treatment with estradiol-17beta (E2) resulted in increased activity of an ERE reporter construct, but not an AP-1 element reporter construct. The antiestrogen ICI 182,780 (ICI) exhibited E2 antagonist activity with both ERalpha and ERbeta. Thus, all components were present for E2-dependent transcription from an ERE except ER; however, cells were not competent for E2-dependent transcription mediated through AP-1. In UBF180 cells, E2 treatment increased both ERE and AP-1 reporter activity. ICI exhibited E2 antagonist activity. Treatment with epidermal growth factor resulted in increased ERE reporter activity that was inhibited by ICI, indicative of ligand-independent activation of ER. These data suggest that multiple pathways for ER-mediated gene regulation occur in the developing fetal uterus and that nuclear components necessary for action of both ERalpha and ERbeta are present prior to expression of the receptor.

Activation of protein kinase C by phorbol esters modulates alpha2beta1 integrin on MCF-7 breast cancer cells.

Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer. In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell-substratum adhesion by the breast adenocarcinoma cell line MCF-7. A PKC activator, 12-O-tetradecanoylphorbol-l, 3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval. This enhanced adhesion was mediated by alpha2beta1 integrin, since both anti-alpha2 and anti-beta1 blocking antibodies each completely abrogated the TPA-induced adhesion. FACS analysis determined that TPA treatment does not change the cell surface expression of alpha2beta1 integrin over a 4-h time interval. However, alpha2beta1 levels were increased after 24 h of TPA treatment. Thus, the enhanced avidity of alpha2beta1-dependent cellular adhesion preceded the induction of alpha2beta1 cell surface expression. Northern blot analysis revealed that mRNA levels of both alpha2 and beta1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of alpha2beta1 mRNA preceded that of its cell surface expression. This further suggested that the TPA-induced avidity of alpha2beta1 was independent of increased expression of alpha2beta1. Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of alpha2beta1 integrin expression and of alpha2beta1-mediated cellular adhesion. To identify a possible mechanism by which TPA could be acting to promote the rapid induction of alpha2beta1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3. C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion. Together, these results suggest that PKC can modulate the alpha2beta1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins alpha2 and beta1 and altering the avidity of the alpha2beta1 integrin by a Rho-dependant mechanism.

Possible presence of enhancing antibodies in idiopathic thrombocytopenic purpura.

It is difficult to detect IgG anti-platelet autoantibodies in idiopathic thrombocytopenic purpura (ITP). Recently, it was reported that reactivity with glycoprotein IIb/IIIa was lost when IgG anti-GPIIb/IIIa antibodies from seven ITP patients were digested with pepsin to yield F(ab')2 fragments. These findings suggested that some IgG antiplatelet autoantibodies in ITP may be of low affinity and thus require the presence of 'enhancing' anti-IgG antibodies (i.e. rheumatoid factors, RFs) for detection. To test this hypothesis, we used a phage display technique to isolate five IgG RFs from an ITP patient (patient 1). Sequence analysis revealed that these RFs consisted of two clones, represented by GG3 and GG48. Both representative RFs bound specifically to IgG Fc fragments with apparent dissociation constants of 8.2 x 10(-8) M and 8.8 x 10(-7) M, respectively. Moreover, IgG RFs were subsequently found in a serum sample from patient 1. Combined, these results suggest that IgG RFs may occur in ITP, and may be required for the detection of some IgG anti-platelet autoantibodies and for the corresponding antibody-mediated platelet destruction in autoimmune ITP.

Expression of estrogen receptor and maintenance of hormone-responsive phenotype in bovine fetal uterine cells.

Objectives were to establish conditions for preparation, growth, and maintenance of a primary culture cell model of fetal uterine cells, and to determine whether cells maintained under those conditions would maintain their capacity to respond to estrogen stimulation in vitro. Fetal uteri (n = 19) were enzymatically dispersed and grown on Type 1 collagen in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum. Fetal-uterine cells appeared fibroblast-like and exhibited positive immunostaining for both vimentin and cytokeratin. Effects of gestational age (GA), passage number (p), and hormonal treatment on appearance of specific mRNAs were determined by RT-PCR; relative concentrations of products determined by densitometry were analyzed as the ratio of target cDNA to the GAPDH loading control. Cells expressed mRNAs for estrogen receptor (ER), TGF-beta, EGF-R, PRL-R, IL-1 alpha, and IL-6. ER mRNA was greater at 185-200 than at 100-110 d GA (P < 0.01). All specific mRNAs examined were greater in p5 cells than p2 at both 100-110 (P < 0.01) and 185-200 d GA (P < 0.02). There was no effect of estradiol on these specific mRNAs in cells from 100-110 d GA; at 185-200 d GA, there was an estradiol (1.0 nM) effect both at 6 hr (P < 0.001) and 24 hr (P < 0.02). Overall, there was an effect of 8-br-cAMP (1 mM; 6 h) on specific mRNAs in cells at both 100-110 (P < 0.001) and 185-200 d GA (P < 0.001). In p5 cells from Day 185-200 GA, there was increased cell proliferation (P < 0.001) in response to estradiol (1 nM; 24 hr). These data suggest that primary fetal uterine cells retain their age-specific and hormone-responsive phenotype under these in vitro conditions.

Development of oestrogen receptor expression and hormone response in the uterus of the bovine fetus.

The objective of the present study was to determine the temporal expression of oestrogen receptor alpha in the uterus of the developing bovine fetus. Bovine fetuses were collected and approximate gestational age was determined by crown-rump measurement. Fetal uteri were either snap frozen in dry ice-ethanol, or placed in sterile Dulbecco's modified Eagle's medium. Fetal uteri (n = 20) were homogenized and cytosolic oestrogen receptor measured by [3H]ligand binding assay. Total RNA was extracted from fetal uteri (n = 53) and amplified by reverse transcription-polymerase chain reaction using primers specific for the oestrogen receptor, progesterone receptor, interleukin 1 alpha, interleukin 6, transforming growth factor beta, prolactin receptor, epidermal growth factor receptor, retinoic acid receptor isoforms alpha, beta, and gamma, or glyceraldehyde-3-phosphate dehydrogenase (loading control). Expressed as a ratio with glyceraldehyde-3-phosphate dehydrogenase, mRNA encoding oestrogen receptor was identified in fetal uteri throughout the period from day 65 to day 200, and was increased from day 100 to day 185 (P < 0.003); uterine samples from day 100 to day 200 expressed interleukin 1 alpha, interleukin 6, transforming growth factor beta, prolactin receptor, epidermal growth factor receptor and retinoic acid receptor isoforms alpha, beta, and gamma, but did not express detectable mRNA encoding progesterone receptor. Despite the presence of mRNA encoding oestrogen receptor, [3H]oestradiol binding was not detected until after day 155. Fetal uterine explants collected at days 100-110 (n = 3) or at days 185-200 (n = 3) were cultured in the presence of oestradiol (1.0 nmol l-1, or vehicle); there was a significant effect of oestradiol treatment on specific mRNA expression at days 185-200 (P < 0.014), but not at days 100-110 (P = 0.71). It is concluded that mRNA encoding oestrogen receptor is constitutively expressed at least from day 65 in the uterus of the bovine fetus, but that oestrogen receptor alpha protein and a functional oestrogen response are not present until late in the second third of pregnancy.